Project Summary Endothelial barrier dysfunction is a central factor in the pathogenesis of protein-rich lung edema and lung inflammation, the hallmarks of acute respiratory distress syndrome (ARDS). However, attempts to develop therapies against crucial element of the disease involving severe disruption of lung vascular endothelial barrier and the progression of protein-rich edema formation thus far have not been successful. The endothelial cell- expressed G-protein coupled receptor sphingosine-1-phosphate receptor-1 (S1PR1), upon ligation by the bioactive lipid sphingosine-1-phosphate (S1P) is an important lung vascular barrier protective mechanism at the level of adherens junctions (AJs). In following the dynamics of cell surface expression of S1PR1, we have made the key observations that S1PR1 expression was decreased during onset of lung vascular injury by endotoxin on the basis of phosphorylation at Tyr143-induced internalization of the receptor. S1PR1 was however re-synthesized resulting in its re-localization to the cell surface during the resolution phase of vascular injury. These temporal findings suggest that re-establishing the pool of cell surface S1PR1 may be crucial for effectiveness of S1P in repairing the lung endothelial barrier. In addition, we observed that S1PR1 once at the cell surface interacted with vascular endothelial growth factor receptor 2 (VEGFR2), suggesting that this interaction may also play an important role in the endothelial barrier re-annealing function of S1P. These observations raise fundamental questions about how S1PR1 is re-synthetized, means by which its cell surface retention is regulated, and the importance of S1PR1 interaction with VEGFR2 in regulating the repair of the lung vascular barrier. Thus, in Project 3 we will pursue the following Specific Aims: (i) we will determine transcriptional regulation of S1PR1 synthesis, specifically the role of the transcription factor Sox2 in regulating endothelial barrier repair and resolution of lung vascular injury; (ii) we will investigate the role of the tyrosine phosphatase SHP-1 in regulating dephosphorylation of the crucial Tyr143 phospho- switch and its involvement in mediating endothelial cell surface retention and responsiveness of S1PR1; and (iii) we will define the function of S1PR1/VEGFR2 interaction as a key mechanism for restoring lung vascular integrity secondary to the stabilization of VE-cadherin at AJs. We will apply a range of powerful approaches such as several novel genetic mouse models (e.g., S1PR1 activity reporter mice), engineered probes, biophysical analysis of AJs, two-photon imaging analysis, and measurements of lung vascular permeability in mice to investigate the novel concept that de novo synthesis and cell surface retention of S1PR1 and S1PR1 interaction with VEGFR2 are required for regulating the lung endothelial barrier repair function of S1P. With these comprehensive and mechanistic studies and through important interactions with the other Projects, we will define the mechanisms regulating endothelial cell S1PR1 function in resolving inflammatory lung vascular injury. We expect our studies to lay new conceptual groundwork for the development of novel therapeutic approaches exploiting S1PR1 signaling as a key target for the treatment of ARDS.